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Interaction of ovalbumin and its asparaginyl-carbohydrate fractions with concanavalin A
356
Biochimica et Biophysica Acta,
535 (1978) 356--369 Biomedical Press
© Elsevier/North-Holland
BBA 37964
INTERACTION OF OVALBUMIN AND ITS ASPARAGINYL-CARBOHYDRATE FRACTIONS WITH CONCANAVALIN A *
VIRGINIA
SHEPHERD
** and REX MONTGOMERY
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 (U.S.A.) (Received
December
20th,
1977)
Summary The interaction of ovalbumin and its asparaginyl-carbohydrate fractions with concanavalin A was studied. Relative affinities were obtained by competitive binding studies using p-nitrophenyl a-D-mannopyranoside. Ovalbumin was separated into two fractions, I and II, by chromatography on concanavalin A-Sepharose. Ovalbumin and its fractions I and II interacted with concanavalin A in solution with binding affinities at 10°C of 2 . l 0 s M -1, 3 - 1 0 4 M-1 and 2 . 1 0 6 M -1, respectively. The seven asparaginyl-carbohydrate fractions, obtained by fractionation on Dowex 50W-X2 (H +) and Durrum DA-4 (borate) column.s, bound to concanavalin A with approximately the same affinity as native ovalbumin, suggesting that the sugar residues for binding in the isolated carbohydrates are exposed in the native protein. The binding of ovalbumin to concanavalin A was minimal after treatment with a-D-mannosidase in spite of the fact that only one half of the available mannose residues were hydrolyzed when compared to those removed by similar treatment of the asparaginylcarbohydrate before fractionation. It is concluded that those a-D-mannosyl residues in ovalbumin that are required for binding to concanavalin A are accessible to a-D-mannosidase while the residual mannosyl groups are " b u r i e d " from interaction with concanavalin A and the enzyme.
* P r e l i m i n a r y c o m m u n i c a t i o n ( 1 9 7 6 ) Fed. P r o c . 35, 1 4 1 9 . ** S u b m i t t e d in D e c e m b e r , 1 9 7 5 , to t h e G r a d u a t e College, U n i v e r s i t y of I o w a , b y V. S h e p h e r d in p a r t i a l f u l f i l l m e n t of t h e r e q u i r e m e n t s f o r t h e d e g r e e of P h . D . A b b r e v i a t i o n s used are: AC, m i x t u r e of L - f l - a s p a r t a m i d o - c a r b o h y d r a t e c o m p o n e n t s , f r e e d f r o m p e p t i d e c o n t a m i n a n t s , isolated f r o m n a t i v e o v a l b u m i n , w i t h e a c h o f t h e original five f r a c t i o n s b e i n g r e p r e s e n t e d AC-A t h r o u g h AC-E. S u b f r a c t i o n s of t h e s e f r a c t i o n s are i d e n t i f i e d b y subscripts, s u c h as AC-C 1 ; d e r i v a t i v e s o b t a i n e d b y r e m o v i n g a - D - m a n n o p y r a n o s y l residues w i t h ¢x-D-mannosidase eaxry a superc r i p t Man, for e x a m p l e Oval M a n a n d AC-D Man. All o t h e r a b b r e v i a t i o n s i n c l u d i n g b i n d i n g p a r a m e t e r s are d e f i n e d in the t e x t .
357 Introduction Concanavalin A, a plant lectin isolated from the jack bean, is widely used in studies of cell surfaces. It exhibits a number of effects on cells upon binding to specific receptors in the cell membrane. These include agglutination of erythrocytes, mitogenesis of lymphocytes, and the differential agglutinability of normal and transformed cells [1]. Some of the binding of concanavalin A is through specific carbohydrate structures in the membrane receptors since the effects can be inhibited by appropriate specific sugars [1]. Concanavalin A binding to glycoproteins and glycopeptides appears to be more complex than simple binding to terminal a-D-mannosyl residues. Goldstein et al. [2] reported that concanavalin A will also bind internal, 1,2-1inked a-Dmannopyranosyl residues with a slightly greater affinity than methyl a-D-mannopyranoside. Furthermore, when internal 1,2-1inked a-D-mannopyranosyl residues occur in sequence, they appear to be more effective than when they occur alone. In addition, a non-carbohydrate, apolar binding site has been identified from X-ray crystallographic data [3,4] and binding studies [5], and Davey et al. have reported that human interferon binds to concanavalin A-Sepharose through hydrophobic interactions [6]. It has been postulated that the b o u n d proteins undergo a rearrangement in structure such that non-specific binding may occur concomitant with sugar interactions [7]. The binding of concanavalin A to receptor molecules in cell membranes, therefore, is n o t only dependent on the presence or absence of mannose in the surface carbohydrate groups but also on non-specific binding sites and the availability or " e x p o s u r e " of the mannose for interaction with the concanavalin A molecule. This paper reports a study of the interaction of concanavalin A with a model glycoprotein, ovalbumin. Comparisons are made of the binding of native ovalbumin and its isolated asparaginyl-carbohydrate to concanavalin A, both free and attached to Sepharose. Materials and Methods Materials Ovalbumin was prepared according to the method of Kekwick and Cannan [8]. Asparaginyl-carbohydrate fractions were isolated according to Huang et al. [9]. The AC-C and AC-D fractions were further subfractionated into AC-CI and -C2, and AC-D~ and -D2 using anion-exchange chromatography in borate buffer [10, 11]. p-Nitrophenyl a-D-mannoside, used for binding experiments, was purchased from P-L Biochemicals and used without further purification. Sugars and other sugar derivatives were purchased from Pfanstiehl. Concanavalin A was prepared by a modification of the m e t h o d reported by Agrawal and Goldstein [12]. Jack bean meal (ICN; 100 g) was extracted overnight with 400 ml of 1.0 M NaC1 at 4°C. The mixture was centrifuged at 8500 rev./min for 15 min at 4°C and the supernatant filtered through cheesecloth. The clear supernatant (approximately 300 ml) was chromatographed on a column (8.5 × 45 cm) of Sephadex G-50 equilibrated with 1 M NaC1. The column was washed with 1 M NaC1 until no more protein could be detected by
358 absorbance at 280 nm. Concanavalin A was eluted with 1 M NaC1 solution containing 0.1 M D-glucose. The solution of concanavalin A was concentrated against PVP to a final protein concentration of approximately 2.5 mg/ml, dialyzed extensively against 0.15 M NaC1, and stored in solution at 5°C. Ovalbumin was fractionated on concanavalin A-Sepharose as previously described [ 11 ].
Immobilization o f concanavalin A Concanavalin A was covalently attached to Sepharose according to the procedure of Porath et al. [13]. Protein concentration on the gel was estimated as 5 mg of concanavalin A per ml of gel by calculating the difference between the amount of concanavalin A in the reaction initially and the uncoupled protein recovered in the wash. The concanavalin A-Sepharose was stored at 4°C in a 0.01 M sodium acetate buffer, pH 5.0, containing 0.15 M NaC1, 1 mM CaC12 and MnCI2, and 0.1% Thimerasol. Identification o f asparaginyl-carbohydrate components o f the ovalbumin fractions U n b o u n d and bound fractions of ovalbumin from concanavalin A-Sepharose chromatography were each digested with pronase and the resulting AC mixtures chromatographed on Dowex 50W-X2 according to Huang et al. [9]. The AC mixtures and the purified, desalted AC-C and AC-D fractions were further chromatographed using anion-exchange chromatography in borate buffer [10, 11]. Digestion o f ovalbumin and A C fractions with a-D-mannosidase-Sepharose Native ovalbumin and its fractions I and II from concanavalin A-Sepharose chromatography, and AC-D were digested with a-D-mannosidase-Sepharose as described by Shepherd and Montgomery [14]. The hydrolyzed mannose was determined using anion-exchange chromatography in borate buffer as described by Lee et al. [10]. Determination o f relative binding capacities o f ovalbumin and AC for concanavalin A at 10°C Relative binding capacities for concanavalin A of ovalbumin and its AC components were determined using inhibition of binding of p-nitrophenyl ~-D-mannoside as described by Bessler et al. [15]. The general procedure involved displacement of a constant known concentration of the chromophore from the binding site on concanavalin A by various concentrations of competing ligands. The release of chromophore was measured spectrophotometrically and binding constants calculated (see Results). All experiments were run in 0.01 M sodium acetate buffer, pH 5.0, with 0.15 M NaC1, 1.0 mM MnC12, and 1.0 mM CaC12, using a Cary 118 recording spectrophotometer with a thermostated cell compartment. Matched divided cells from Hellma with a path length of 8.75 mm were used. Ligands tested for competition with p-nitrophenyl a-D-mannoside were the following: ovalbumin; ovalbumin fractions I and II; ovalbumin treated with a-D-mannosidase (OvalMan); ovalbumin fraction II treated with ~-D-mannosidase (Oval II Man ); AC-A, -B, -C, -C1, -C2,
359
-D, -D1, -D2 and -E; AC-D treated with a - D - m a n n o s i d a s e (AC-DMan); and methyl a-D-mannoside. Concentrations of ovalbumin and its derivatives were deter'e 280nm 1~ = 7.5) and by the phenol/sulfuric acid mined spectrophotometrically ~ m e t h o d [16] for mannose equivalents. All other ligand concentrations were determined by weight and by quantitation of mannose equivalents. Essentially, the only modifications in the m e t h o d of Bessler et al. [15] were the following: (a) the concentration of p-nitrophenyl a-D-mannoside in the cuvette after mixing was 0.05 mM in displacement experiments with competing ligands, and (b) the pH and composition of the buffer was as described above.
Determination o f binding affinities o f ovalbumin and A C-D for concanavalin A at 25°C A comparison of K D values was made for ovalbumin and AC-D binding to concanavalin A at 25°C by the m e t h o d described above at 10°C based on the model by Bessler et al. (using displacement of a constant a m o u n t of p-nitrophenyl ~-D-mannoside) and by the following procedure (using displacement of a series of concentrations of p-nitrophenyl a-D-mannoside by a fixed concentration of competing ligand): a stock solution o f p - n i t r o p h e n y l a-D-mannoside of known concentration (approximately 0.5 mM) was prepared. Aliquots of this solution were pipetted into tubes and buffer was added to a final volume of 2.7 ml, giving final concentrations of p-nitrophenyl a-D-mannoside of 0.05 to 0.25 mM. To each of these solutions was added 0.3 ml of a known concentration of a ligand solution. A series of these experiments.was set up [15], resulting in a set of displacement curves at variable chromophore concentrations and a constant a m o u n t of total ligand. Difference spectra were recorded at 25°C using a Perkin-Elmer 124 recording spectrophotometer. Double reciprocal plots of [PT]/[PD] versus 1/[D] were constructed at each ligand concentration as described by Klotz [17]. The slope of this line is 1/Kapp where KD KaPp - KL[L] + 1 " K D is the association constant o f p - n i t r o p h e n y l a-D-mannoside for concanavalin A, K L is the ligand association constant and [L] is ligand concentration.
Results
Ovalbumin can be separated into two components on concanavalin A-Sepharose [11] to give an u n b o u n d fraction (I), and a bound fraction (II) which was eluted with 0.1 M m e t h y l a-D-mannoside or -glucoside [11]. The ratio of II to I was 2 : 1 with an overall recovery of 85--90%. Ovalbumin fractions I and II contained 4.2 and 2.3 mol of glucosamine per mole of protein, respectively. The binding of ovalbumin fraction II occurred only below 15°C. However, once bound, fraction II could n o t be removed by raising the temperature. The effects of the differing carbohydrate structures on binding were studied by digesting the ovalbumin fractions I and II four times with pronase to obtain the corresponding AC fractions as described previously [9,11]. Fraction II contained primarily AC-C2, -D2, and -E with some AC-A present. Fraction I contained AC-B, -C~, and -D1, again with some AC-A. Ovalbumin fraction II
360 could be further subfractionated on the basis of the differing binding affinities of AC-C2, -D2 and -E for concanavalin A. After elution of ovalbumin fraction I, the column of concanavalin A-Sepharose was developed with a linear gradient up to 0.1 M methyl a-D-glucoside. No definitive separation of peaks occurred, but it was evident that more than one component was present, and that the AC-A, -C2, -D2 and -E fractions in the whole protein were further fractionated.
Determination of relative binding affinities of ovalbumin and AC for concanavalin A Although the concanavalin A-Sepharose column was useful in the study of ovalbumin binding to free concanavalin A and subsequent fractionation of the protein on the basis of microheterogeneity in the carbohydrate, some difficulty was encountered in the study of AC binding to concanavalin A-Sepharose because AC-C2, -D2 and-E were n o t eluted with 0.1 M methyl-a-D-mannoside. It was therefore necessary to use a different technique to compare the relative binding affinities of the whole protein and its isolated carbohydrate for concanavalin A. Bessler et al. [15] described a spectroscopic method for the determination of binding constants for various simple sugar ligands to concanavalin A. This m e t h o d utilizes the blue shift seen when p-nitrophenyl a-D-mannoside binds to protein [18], with a m a x i m u m absorbance change at 317 nm. With certain systems the amounts of free and bound chromophore, [D] and [PD], free and bound ligand, [L] and [PL], and free protein [P] can be calculated from the observed absorbance change, and binding constants for the competing ligands can be determined. The equilibria are represented below: L + P + D.K~PD + L
PL + D.
~PDL
where KD is the association constant for p-nitrophenyl a-D-mannoside, KL is the association constant for the ligand L, KLD is the association constant for the ligand L for the PL complex, and KDL is the association constant for the ligand L for the PD complex. The expression of mass balance (2) can be written: [PT] = [PD] + [P] + [PL] + [PDL]
(2)
Relationship 3 can then be derived from 1 and 2: I
[P_T] i _ 1] = 1 + Kb[L] [PD] + [PDL] J [D] KD[1 +KDL[L]
(3)
By defining the concentrations of free and complexed p-nitrophenyl a-D-mannoside in Eqn. 3 in terms of the experimental data (see Materials and Methods and ref. 15) Eqn. 4 can be written:
I AAmax
~kA
1 + EL[L]
361 For the determination of the value of KDL, data for each value of [L] (approximated by [LT]) were fit by a non-linear least squares program (Nonlin, C.M. Metzler, The Upjohn Company), using values for K L between 120 and 1000 mM-1. According to Bessler et al. [15] the binding of simple sugars to concanavalin A can be represented by a system where only one molecule can bind at a carbohydrate site on the concanavalin A monomer. This assumption eliminates the [PDL] complex, KDL and KLD become very small and Eqn. 3 reduces to Eqn 5: [F[PDI[PT]
11
[D] = 1/KD + ~ [ L ]
(5)
A plot of ([PT]/[PD] -- 1) [D] versus [L], where [L] is approximated by [LT] , should give a straight line with a slope equal to KL/K D and a y-intercept equal to 1/K D. The assumption that [L] = [LT] Can be made when [PL] < < [LT]. Eqn. 5 can be rearranged to form Eqn. 6 in which all the terms on the right side of the equation are constants or can be experimentally determined, and a value for E L can be calculated at each total ligand concentration. [PD] [PT] [PD] KD[D]
KL=
[PD] F
KD[D]L[LT] -- [Pw] +
KD[D-----~[PD] + [PD]]
(6)
This equation allows for the determination of g L without approximating [L] to [LT]. The relative binding affinities of ovalbumin and its AC fractions were compared as competing ligands using Eqns. 4, 5 or 6.
Inhibition by several ovalbumin fractions o f binding o f p-nitrophenyl a-D-mannoside to concanavalin A It was found both in this study and by Bessler et al. [15] that the evaluation of binding constants assuming one binding site on each concanavalin A m o n o m e r is valid for methyl a-D-mannopyranoside, for which a linear plot results. The binding constant so calculated in the present study for this ligand was 1.7 • 104 M -1 at pH 5.0 and 10°C, compared to 1.65 • 104 M -1 at pH 7.0 and 10°C [15]. For methyl a-D-mannoside and other ligands where concentrations necessary for binding are large and binding constants are small, the total a m o u n t of ligand b o u n d is small compared to the total ligand present, and indeed [L] can be approximated by [LT]. Ovalbumin fractions I and II, Oval Man and Oval II Man were used as competing ligands for the binding of p-nitrophenyl a-D-mannoside to concanavalin A at 10°C and pH 5.0. The plots of ([PT]/[PD] -- 1)[D] versus [LT] were linear over the concentrations studied and the fractions I, II and Oval Man showed binding constants of 3.0 • 104 M -1, 2.0 • 106 M -1 and 1.7 • 104 M -I, respectively (Fig. 1). Oval II Man did n o t displace p-nitrophenyl a-D-mannoside from concanavalin A over these same concentrations. The competitive binding data for the native ovalbumin did n o t show a linear relationship when plotted according to the relationship of Eqns. 4, 5 or 6, so
362
0.4--
E
-
a ~
°.2-i
121
O--
°.~J--
OvalTr
i
o/250
/
m
Q
VO.2~
;o°
D
X
X
O~al
0.1-Q
X
v
O-J
0'.2
[LT] (mM)
I
°.'04
I
I 0.02
I
I 0.0,
[LT] (mM)
Fig. 1. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] versus [ L T ] f o r o v a l b u m i n (Oval), o v a l b u m i n f r a c t i o n I ( O v a l I), ovalb u m i n f r a c t i o n II ( O v a l II) a n d o v a l b u m i n t r e a t e d w i t h c~-D-mannoside ( O v a l M) a t 1 0 ° C , a c c o r d i n g t o E q n . 5. T h e t o t a l c o n c e n t r a t i o n o f c o n c a n a v a l i n A ( [ P T ] ) is 0 . 0 3 9 m M in c o m b i n i n g sites, a s s u m i n g a m o n o m e r m o l e c u l a r w e i g h t o f 2 7 0 0 0 . T h e p - n i t r o p h e n y l c~-D-mannoside c o n c e n t r a t i o n is 0 . 0 5 m M a f t e r m i x i n g . O t h e r e x p e r i m e n t a l d e t a i l s are d e s c r i b e d u n d e r M e t h o d s . Fig. 2. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T] f o r o v a l b u m i n a t 2 5 a n d 1 0 ° C . O t h e r c o n d i t i o n s are as d e s c r i b e d in Fig. 1 a n d u n d e r M e t h o d s .
that these three models did not account for the complexity. However, the possibility that there is cooperative interactions of the binding of ovalbumin molecules at the two sites of a concanavalin A dimer is suggested by the linear plot of ([PT]/[PD] -- 1)[D] against [LT] 2 It was known from studies of the binding of ovalbumin to concanavalin A-Sepharose, that negligible interaction occurred at 25°C. The interaction of ovalbumin with concanavalin A in solution was studied at 25°C to determine if the complexity of binding at 10°C could be eliminated by an increase in temperature. The experiments were conducted as described for 10°C. A plot of ( [ P T ] / [ P D ] - 1)[D] versus [LT] could be approximated by a straight line (Fig. 2), and from the slope of this line, a binding constant for ovalbumin at 25°C was calculated to be 1.7 • 10 s M -1. Experiments were also conducted at 25°C in which p-nitrophenyl ~-D-mannoside at differing concentrations was displaced from the concanavalin A by a fixed a m o u n t of ovalbumin. The results were analyzed using the double reciprocal plot described by Klotz [17]. A linear relationship was found at concentrations of ovalbumin up to 0.0158 mM. Ligand binding constants were calculated at each concentration with the following results: at [ L T ] ---- 0.0026 mM, E L = 1 . 5 " l 0 s M - l ; at [ L T ] = 0.0079 mM, K b = 1.6 • 10 s M-'; and, a t [ L T ] = 0 . 0 1 5 8 mM, K L = 0 . 8 " l 0 s M - 1 . T h e plot at low concentrations of ovalbumin gave a y-intercept of n = 1, indicating competitive displacement of p-nitrophenyl ~-D-mannoside from the
363
TABLE I A SUMMARY OF THE BINDING C O N S T A N T S FOR O V A L B U M I N AND ITS D E R I V A T I V E S Ligand
Temp.
K L × 10 -5 (M - I )
(°C)
Method of calculation * 1
Ovalbumin Ovalbumin fraction I O v a l b u m i n f r a c t i o n II Oval Man
10 25 10 10 10
2
3
1.6±0.8 1.70 0.30 20.00 0.17
1.23 -+ 0 . 1 1
* 1~ E q n . 5; 2, eqn. 6; 3, d o u b l e r e c i p r o c a l p l o t s o f [ P T ] / [ P D ] against 1 / [ D ] .
carbohydrate binding site and one to one binding to concanavalin A. The Scatchard plots [19] at low protein concentrations were linear, but plots at higher concentrations (>0.0079 mM) showed deviations from linearity, suggesting that some cooperativity is present also at 25°C. A summary of the binding constants determined for all the ovalbumin fractions examined is shown in Table I.
Inhibition by the AC fractions o f binding p-nitrophenyl a-D-mannoside to concanavalin A The interaction of the isolated ovalbumin AC fractions with concanavalin A was investigated to compare the relative binding affitdties of the AC to the whole protein and to determine which units of the carbohydrate structure were important for binding. The five AC fractions isolated from Dowex-50 chromatography were used as competitive inhibitors of the binding of p-nitrophenyl a-D-mannoside to concanavalin A at 10°C (Fig. 3). As in the case of ovalbumin, all the plots were non-linear over approximately the same concentration range as that used for ovalbumin. Attempts to linearize the data using the models expressed in Eqns. 4, 5 and 6 were again unsuccessful. The use of Eqn. 6 for AC-D, to calculate KL at each total ligand concentration, resulted in values for KL which increased with increasing [LT], ranging from 6.6 • 104 M -1 at [LT] = 0.013 mM, to 1 . 0 . 106M -' at [LT] = 0.038 mM. However, plots of ( [ P T ] / [ P D ] - - 1 ) [ D ] versus [LT] 2 were linear, suggesting some cooperative interaction of the binding of the AC molecules to each unit in the concanavalin A dimer. Fig. 4 shows the results of the binding of AC-D, -D1, -Df, and -D Man to concanavalin A at 10°C. Fig. 5 shows similar plots for the subfractions AC-C, and -C2. AC-D Man and AC-C1 have almost no affinity for concanavalin A, while AC-D2 and AC-C2 are both slightly stronger in their binding than the parent AC-D and -C fractions. At 25°C, as in the case of ovalbumin, the binding of AC-D to concanavalin A became less complex, and plots of ([PT]/[PD] -- 1)[D] versus [LT] (Fig. 6) and the double reciprocal plots were linear. The binding constant from Figure 6 was calculated at 0.7 • 10 s M -1. Binding constants from the double reciprocal plots were as follows: at [LT] = 0.014 raM, KL = 0.6 • l 0 s M-l; at [LT] = 0.028 mM.
364
0.6--
I.S--
D
E
A C
~
~ O , 4 - -
a
q
!
o
°0--
.i o
t"7 o.s-
t"7 o . 2 - O_
13
0._
•t- ×
D1 0.0--
x
0-0--
1 0.00
[
0.02
[LTI
I
l
l
0-04
I
O.OG
I
0-0
(mM)
I
I
0.I
7-
0.2
ILT] (mM] .
.
o
Fig. 3. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T ] f o r t h e a s p a r a g m y l - c a x b o h y d r a t e f r a c t i o n s a t 10 C. O t h e r c o n d i t i o n s axe as d e s c r i b e d in Fig. 1 a n d u n d e r M e t h o d s . Fig. 4. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T ] f o r t h e A C - D s u b f r a c t i o n s at 1 0 ° C . O t h e r c o n d i t i o n s axe as d e s c r i b e d in F i g . I a n d u n d e r M e t h o d s .
0.6--
0.4--
C
E
E 0*4--
r7
~--
J
0.2--
i i
r7
i2s°
0-2--
O. q
0.0--
0.0--
I
0.00
I
I
0.02
[LT]
I
I
0.04
(mM)
I
I
0-08
0.0
I
I
0.02
1
[
0.04
[LT] (raM)
F i g . 5. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [L T] f o r t h e AC-C s u b f r a c t i o n s a t 1 0 ° C . O t h e r c o n d i t i o n s axe as d e s c r i b e d in F i g . 1 a n d u n d e r M e t h o d s . F i g . 6. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T ] f o r t h e A C - D s u b f r a c t i o n s a t 2 5 a n d I O ° C . O t h e r c o n d i t i o n s a t e as d e s c r i b e d in F i g . 1 a n d u n d e r M e t h o d s .
365 TABLE
II
A SUMMARY
Ligand
AC-D AC-D]:, A C - D lvian
OF THE BINDING
CONSTANTS
Temp. (°C)
25 10 10
FOR
THE AC-D FRACTIONS
K L X I0 -s ( M -i) M e t h o d of calculation *
1
2
3
0.7 0.25 0.07
1.1±0.2
0.52±0.18
* 1, E q n . 5; 2, e q n . 6; 3, d o u b l e r e c i p r o c a l p l o t s o f [ P T ] / [ P D ]
against 1/[D].
K L = 0.3 • 10 s M-l; and, at [LT] = 0.043 mM, K L = 0.7 • 10 s M -1. Using Eqn. 6 the binding co n s tant at [aT] = 0.028 mM was calculated as 0.9 ± 0.2 • 10 s M-'; and, at [LT] = 0.43, K L = 1.3 ± 0.1 • 10 s M -1. A summary of the binding constants for the AC-D fractions is shown in Table II. Discussion Concanavalin A, covalently attached to Sepharose, has been used as a convenient m e t h o d for studying the interaction o f macromolecules with this lectin. In most instances, the b o u n d protein is eluted from the concanavalin A-Sepharose using 0.1 M m e t h y l a-D-mannoside or -glucoside. Native ovalbumin was separated into two fractions, one which elutes with starting buffer (I), and the o th e r (II) that is eluted with 0.1 M m et hyl a-D-glucoside or -mannoside [ 11]. F u r t h e r fractionation o f fraction II was achieved using a linear gradient o f m e t h y l a-D-mannoside, indicating a heterogeneity of binding species. As will be shown later, the fractions can be related to the microheterogeneity of the c a r b o h y d r a t e groups in the glycoprotein and their differing affinities for concanavalin A. The c a r b o h y d r a t e of ovalbumin has been separated into seven asparaginyl c a r b o h y d r a t e fractions, some of which m ay still be heterogeneous [11]. These fractions may be grouped into three classes: Class I with no non-reducing terminal a-D-mannosyl residues, Class II with bot h a-D-mannosyl and N-acetyl~-D-glucosaminyl non-reducing terminal residues, and Class III with only a-D-mannosyl non-reducing terminal residues. By pronase digestion of the ovalbumin fractions I and II and subsequent isolation of the AC-fractions [11] it was shown that fraction I contained c a r b o h y d r a t e groups that were principally of Class I and fraction II principally of Class III, with some overlap of the Class II c a r b o h y d r a t e groups. Since it was required to com pa r e the binding of ovalbumin and the AC-fractions to concanavalin A it was impossible to use equilibrium dialysis or a concanavalin A-Sepharose column, to which ovalbumin fraction I did n o t bind. A spectroscopic p r o c e d u r e of Bessler et al. [15] was followed in which the competition for the binding to concanavalin A o f p - n i t r o p h e n y l a-D-mannoside and the c a r b o h y d r a t e ligand in question was determined from the difference in the spectrum o f b o u n d and free p - n i t r o p h e n y l a-D-mannoside. The model of Bessler et al. [15] assumes one t o one binding at the c a r b o h y d r a t e site, and for
366 simple sugars, such as methyl a-D-mannoside, this model is valid. However, other types of interactions cannot be ruled out by this procedure and indeed ovalbumin and its AC fractions showed non-linear binding, indicating a greater complexity than the simple model that applied to methyl a-D-mannopyranoside. Several workers have suggested that other non-carbohydrate interactions might be involved in the binding of ligands to concanavalin A. Human interferon apparently binds to concanavalin A-Sepharose through both carbohydrate and non-polar interactions [6], and binding of concanavalin A to fat cells is through multiple binding sites, stabilized by both carbohydrate and hydrophobic forces [ 7]. The competitive inhibition data for ovalbumin and its AC fractions when expressed assuming the model of Bessler showed an initial slope that progressively increased as the competing ligand concentration increased. In those cases where there was a high affinity binding, such as AC-D2 or ovalbumin fraction II the lower affinity at small ligand concentrations was not measurable or was less apparent. Similarly, under the conditions of the experiment, ligands with little or no affinity for concanavalin A, such as ovalbumin fraction I, AC-C1, or AC-D~ do not show the non-linear plot. Ovalbumin, containing a mixture of at least seven glycoproteins, presents a curved plot on this Bessler model, as a result of the two extremes of binding. The curvature in the plot (Fig. 1) is probably n o t simply the result of a mixture of binding affinities at the carbohydrate site since presumably the tightest binding component would bind first, giving a plot curving in the opposite manner to that observed. Additionally, it is clear from the change in binding with concanavalin A at two temperatures, 10 and 25°C, that the conformations of the proteins, aside from the carbohydrate ligand, play a role in the nature of the complexation. Since the spectroscopic procedure of Bessler et al. can only detect the competitive displacement of the p-nitrophenyl a-D-mannoside from the carbohydrate binding site on each m o n o m e r unit, it is only by indirect evidence that non-carbohydrate binding or cooperative interactions are indicated. Such is the case however for ovalbumin and its AC fractions since the data show a linear relationship between ( [PT] / [ PD] -- 1) [D] and [ L w ] 2. A more detailed description of this cooperative binding at the two sites of the concanavalin A dimer requires a different experimental approach, such as equilibrium dialysis for the AC fractions, and the means to prepare pure glycopeptides, which is presently possible in a relatively few cases. Although quantitative binding constants were n o t obtained for all of the fractions studied, relative binding affinities could be established on the basis of concentrations of ligand necessary to displace 50% of the bound p-nitrophenyl a-D.mannoside, analogous to the methods of calculation using dextran binding inhibition [20,21], hemagglutination inhibition [22], and inhibition of binding of '2SI-concanavalin A to erythrocytes [23]. Although these methods may not result in accurate binding constants, an approximation of relative affinities can be obtained. Table III shows the 50% inhibition concentration for the ovalbumin and AC fractions. On the basis of concentrations of AC fractions necessary for p-nitrophenyl a-D-mannoside displacement, the fractions are collected into four groups from the relative binding affinities: Group 1 (AC-D2 and AC-C2), which has an affinity for concanavalin A approximately 8--9 times
367 TABLE III RELATIVE
BINDING CAPACITIES
Ligand
Ovalbumin Ovalbumin fraction I Ovalbumin fraction II Oval Man
OF OVALBUMIN
Conc. to give
Relative binding
50% i n h i b i t i o n (M × 1 0 - s )
capacity *
3.0
AC.C1 AC-C2 AC-C AC-D 1 AC-D 2 AC-D AC-D Man AC-E M e t h y l cx-D-manno side
TO Con A
5.3 2 . 0 ** 160.0 1 . 2 **
0.1
Oval II Man AC-A AC-B
AND AC FRACTIONS
*** 3.0 4.5
5.3 3.6 *** 8.0 5.3 2 . 5 ** 8.9 8.0 1 . 0 **
2.0 3.0 1.8 2.0 2.2 16.0
7.3 1.0
* R e l a t i v e t o t h e c o n c e n t r a t i o n o f m e t h y l a - D - m a n n o s i d e t o g i v e 5 0 % i n h i b i t i o n = 1.0. ** C a l c u l a t e d f r o m a r a t i o o f KL/KM, w h e r e K M = t h e a s s o c i a t i o n c o n s t a n t f o r m e t h y l - a - D - m a n n o s i d e . ** * F o r t h e c o n c e n t r a t i o n s t u d i e d n o b i n d i n g w a s o b s e r v e d .
greater than m e t h y l a-D-mannoside; Group 2 (AC-A and AC-E) with an affinity 5--7 times greater; Group 3 (AC-B, AC-D1 and AC-D Man) about the same as m e t h y l a-D-mannoside; and, Group 4 (AC-C1) which shows less binding than the monosaccharide. The structures within these fractions that are binding to concanavalin A might be predicted on the basis of model c o m p o u n d studies and known structural features of the AC components [11,24]. Goldstein and co-workers [25,26] have investigated the binding of several mannose-conraining di- and trisaccharides, as shown below relative to methyl a-D-mannoside: methyl a-D-mannoside Gal-a-(1->6)-Man-a-(I->2 )-Man Mama-(1->2 )-Man Gal-a-(1->2 )-Man-a-(I->2 )-Man Man-a-(1-~2 )-Man-a-(I->2 )-Man
1.0 1.2 1.9 2.1 10.0
AC-C1, from which no a-D-mannosyl residues were hydrolyzed by a-D-mannosidase and no c/s-hydroxyl groups on the internal mannose residues complex with borate [11], shows a binding to concanavalin A less than that for methyl a-D-mannopyranoside. AC-B, even with no terminal a-D-mannosyl residues, shows binding equivalent to m e t h y l a-D-mannopyranoside and is probably due to internal 1 -~ 2 linked a-D-mannosyl residues, such as proposed earlier, where AC-B is derived from AC-D2 by the addition of N-acetylglucosaminyl residues on each terminal mannose [11]. This is also consistent with the weak complexation of AC-B with borate [11]. Similarly, the binding of AC-D Man is due to the residual AC-D1, from which only one mannosyl residue is hydrolyzed by a-D-mannosidase [ 11].
368 The tight binding of AC-E and AC-D2 agrees with the fact that each oligosaccharide has three terminal a-D-mannosyl residues, the addition affinity in AC-D2 being due to the internal 1 -~ 2 linked mannosyl residue [24]. The correlation between the complexation with concanavalin A and borate is again striking. The intermediate binding affinity of Group 2 compounds, AC-A and AC-C2, would be predicted by the presence in each of a terminal and a 1 -~ 2 linked a-D-mannosyl residue. The order of relative binding affinities for the ovalbumin fractions from Table III is as follows: Ovalbumin fraction II > ovalbumin > ovalbumin fraction I > O v a l Man ~ Oval II Man. Fraction I, which is not retained on a column of concanavalin A-Sepharose, does bind to concanavalin A in solution; apparently the binding is not tight enough to retard this component on the concanavalin A-Sepharose column. As expected, the ovalbumin fraction II, which binds to immobilized concanavalin A, also binds tightly to concanavalin A in solution. It will be noted (Table III) that ovalbumin fraction II, which contains the carbohydrate groups AC-D2 and AC-E, has a binding capacity that is about 20 times greater than the asparaginyl-carbohydrates. It is proposed that the concanavalin A and ovalbumin proteins interact through non-carbohydrate sites thus statistically increasing the probability of the a-D-mannosyl residues in fraction II binding with the concanavalin A carbohydrate binding site. Competition with the p-nitrophenyl a-D-mannoside would thus appear to be increased over that of the AC-D2 or AC-E, which do not have the same noncarbohydrate interactions. It has been shown [14] that 40% of the total mannose in the isolated AC can be removed enzymatically, but only one-half of this value or 20% of the total mannose residues, can be hydrolyzed from native ovalbumin. Following the hydrolysis of ovalbumin fraction II by a-D-mannosidase, no binding to concanavalin A was observed over the concentrations studied. Apparently, only the peripheral mannose residues in this protein are important for and accessible to interaction with both concanavalin A and a-D-mannosidase, with the core region perhaps " b u r i e d " in a portion of the polypeptide structure. This result implies that the inner " c o r e " region contributes little or no binding in the interaction of the ovalbumin glycopeptide with concanavalin A. Native ovalbumin and its AC fractions bind with approximately the same affinity (Figs. 1 and 3). Thus the carbohydrate residues which interact with concanavalin A are equally exposed in the whole protein and the isolated AC. This is in agreement with the finding of Smith et al. [22] who found ovalbumin and its glycopeptide equally effective in inhibiting hemagglutination by concanavalin A. In contrast, Young and Leon [21] found that the glycopeptide mixture inhibited dextran precipitation at concentrations ten-fold less than the whole protein. In both cases, however, comparisons with the present study are difficult since methods for and conditions of measurement of binding were different. In comparing the results of studies on the binding of ligands to concanavalin A it is important to consider the system being used. Kornfeld and Ferris [23] looked at the inhibition by IgG glycopeptides of the binding of concanavalin A to cell surfaces. This type of binding is very complex [7] and probably involves the binding of concanavalin A to the cells through non-carbohydrate as
369 well as carbohydrate interactions. The binding of concanavalin A to individual proteins and proteins within a membrane is probably much more complex than binding to monosaccharides, as was found in the present study for the ovalbumin-concanavalin A interaction. It will be a matter for future to study to resolve the differences in binding affinities of glycopeptides to concanavalin A and the inhibitory properties of these glycopeptides in the binding of concanavalin A to cell membranes. Acknowledgement This research was supported by Grant GM14013 from the United States Public Health Se~zice. References 1 Reeke, G.N., Becker, J.W., Cunningham, B.A.1 Gunther, G.R., Wang, J.L. and Ede l ma nl G.M. (1974) Ann. N.Y. Acad. Sci, 234, 369--382 2 Goldstein, I.J., Reichert, C.M., Misaki, A. and Gorin, R.A.J. (1973) Biochim. Biophys. Acta 317, 500--504 3 Edelman, G.M., Cunningham, B.A., Reeke, Jr., G.N., Becket, J.W., Waxdal, M.J. and Wang, J.L. (1972) Proe. Natl. Aead. Sei. U.S. 69, 2 5 8 0 - - 2 5 8 4 4 Hardman, K.D. and Ainsworth, C.F. (1972) Biochemistry 12, 4442--4448 5 Loontiens, F.G., Van Wauwe, J.P., Degussen, K, and De Bruyne, C.K. (1973) Carbohydr. Res. 30, 51--62 6 Davey, M.W., Huang, J.W., Sulkowski, E. and Carter, W.A. (1974) J. Biol. Chem. 249, 6354--6355 7 Cuatrecasas, P. (1973) Biochemistry 12, 1312--1323 8 Kekwiek, R.A. and Cannan, R.K. (1936) Biochem. J. 3 0 , 2 2 7 9 Huang, C.-C., Mayer, H. and Mongomery, R. (1970) Carbohydr. Res. 1 3 , 1 2 7 - - 1 3 7 10 Lee, Y.C., Johnson, G.S., White, B. and Scocca, J. (1971) Anal. Biochem. 431 640--641 11 Shepherd, V. and Montgomery, R. (1978) Carbohydr. Res. 6 1 , 1 4 7 - - 1 5 7 12 Agrawal, B.B.L. and Goldstein, I.J. (1965) Biochem. J. 96, 23C--25C 13 Porath, J., Aspberg, K., Drevlin, H. and Axen, R. (1973) J. Chromatogr. 86, 53--56 14 Shepherd, V. and Montgomery, R. (1976) Bioehim. Biophys. Acta 429, 884--894 15 Besslet, W., Shafer, J.A. and Goldstein, I.J. (1974) J. Biol. Chem. 249, 2819--2822 16 Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Anal. Chem. 2 8 , 3 5 0 - - 3 5 6 17 Klotz, I.M. and Hunston, D.L. (1971) Biochemistry 10, 3 065--3069 18 Hassing, G.S. and Goldstein, I.J. (1970) Eur. J. Biochem. 1 6 , 5 4 9 - - 5 5 6 19 Scatchard, G. (1949) Ann. N.Y., Acad. ScL 5 1 , 6 6 0 - - 6 7 2 20 Goldstein, I.J., Hollerman, C.E. and Smith, E.E. (1965) Biochemistry 4, 876--883 21 Youngl N.M. and Leon, M. (1974) Biochim. Biophys. Acta 3 6 5 , 4 1 8 - - 4 2 4 22 Smith, D.F., Neri, G. and Walborg, E.F. (19"/3) Biochemistry 12, 2111--2118 23 Kornfeld, R. and Ferris, C. (1975) J. Biol. Chem. 250, 2 6 1 4 - - 2 6 1 9 24 Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, M., Muramatsu, T., Iwashita, S., Inoue, Y. and Kobata, A. (1975) J. Biol. Chem. 250, 8569--8575 25 So, L.L. and Goldstein, I.J. (1968) J. Biol. Chem. 243, 2003--2007 26 Goldstein I I.J., Reichert, C.M. and Misaki, A. (1974) Ann. N.Y. Acad. Sei. 2341 283--296
Biochimica et Biophysica Acta,
535 (1978) 356--369 Biomedical Press
© Elsevier/North-Holland
BBA 37964
INTERACTION OF OVALBUMIN AND ITS ASPARAGINYL-CARBOHYDRATE FRACTIONS WITH CONCANAVALIN A *
VIRGINIA
SHEPHERD
** and REX MONTGOMERY
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 (U.S.A.) (Received
December
20th,
1977)
Summary The interaction of ovalbumin and its asparaginyl-carbohydrate fractions with concanavalin A was studied. Relative affinities were obtained by competitive binding studies using p-nitrophenyl a-D-mannopyranoside. Ovalbumin was separated into two fractions, I and II, by chromatography on concanavalin A-Sepharose. Ovalbumin and its fractions I and II interacted with concanavalin A in solution with binding affinities at 10°C of 2 . l 0 s M -1, 3 - 1 0 4 M-1 and 2 . 1 0 6 M -1, respectively. The seven asparaginyl-carbohydrate fractions, obtained by fractionation on Dowex 50W-X2 (H +) and Durrum DA-4 (borate) column.s, bound to concanavalin A with approximately the same affinity as native ovalbumin, suggesting that the sugar residues for binding in the isolated carbohydrates are exposed in the native protein. The binding of ovalbumin to concanavalin A was minimal after treatment with a-D-mannosidase in spite of the fact that only one half of the available mannose residues were hydrolyzed when compared to those removed by similar treatment of the asparaginylcarbohydrate before fractionation. It is concluded that those a-D-mannosyl residues in ovalbumin that are required for binding to concanavalin A are accessible to a-D-mannosidase while the residual mannosyl groups are " b u r i e d " from interaction with concanavalin A and the enzyme.
* P r e l i m i n a r y c o m m u n i c a t i o n ( 1 9 7 6 ) Fed. P r o c . 35, 1 4 1 9 . ** S u b m i t t e d in D e c e m b e r , 1 9 7 5 , to t h e G r a d u a t e College, U n i v e r s i t y of I o w a , b y V. S h e p h e r d in p a r t i a l f u l f i l l m e n t of t h e r e q u i r e m e n t s f o r t h e d e g r e e of P h . D . A b b r e v i a t i o n s used are: AC, m i x t u r e of L - f l - a s p a r t a m i d o - c a r b o h y d r a t e c o m p o n e n t s , f r e e d f r o m p e p t i d e c o n t a m i n a n t s , isolated f r o m n a t i v e o v a l b u m i n , w i t h e a c h o f t h e original five f r a c t i o n s b e i n g r e p r e s e n t e d AC-A t h r o u g h AC-E. S u b f r a c t i o n s of t h e s e f r a c t i o n s are i d e n t i f i e d b y subscripts, s u c h as AC-C 1 ; d e r i v a t i v e s o b t a i n e d b y r e m o v i n g a - D - m a n n o p y r a n o s y l residues w i t h ¢x-D-mannosidase eaxry a superc r i p t Man, for e x a m p l e Oval M a n a n d AC-D Man. All o t h e r a b b r e v i a t i o n s i n c l u d i n g b i n d i n g p a r a m e t e r s are d e f i n e d in the t e x t .
357 Introduction Concanavalin A, a plant lectin isolated from the jack bean, is widely used in studies of cell surfaces. It exhibits a number of effects on cells upon binding to specific receptors in the cell membrane. These include agglutination of erythrocytes, mitogenesis of lymphocytes, and the differential agglutinability of normal and transformed cells [1]. Some of the binding of concanavalin A is through specific carbohydrate structures in the membrane receptors since the effects can be inhibited by appropriate specific sugars [1]. Concanavalin A binding to glycoproteins and glycopeptides appears to be more complex than simple binding to terminal a-D-mannosyl residues. Goldstein et al. [2] reported that concanavalin A will also bind internal, 1,2-1inked a-Dmannopyranosyl residues with a slightly greater affinity than methyl a-D-mannopyranoside. Furthermore, when internal 1,2-1inked a-D-mannopyranosyl residues occur in sequence, they appear to be more effective than when they occur alone. In addition, a non-carbohydrate, apolar binding site has been identified from X-ray crystallographic data [3,4] and binding studies [5], and Davey et al. have reported that human interferon binds to concanavalin A-Sepharose through hydrophobic interactions [6]. It has been postulated that the b o u n d proteins undergo a rearrangement in structure such that non-specific binding may occur concomitant with sugar interactions [7]. The binding of concanavalin A to receptor molecules in cell membranes, therefore, is n o t only dependent on the presence or absence of mannose in the surface carbohydrate groups but also on non-specific binding sites and the availability or " e x p o s u r e " of the mannose for interaction with the concanavalin A molecule. This paper reports a study of the interaction of concanavalin A with a model glycoprotein, ovalbumin. Comparisons are made of the binding of native ovalbumin and its isolated asparaginyl-carbohydrate to concanavalin A, both free and attached to Sepharose. Materials and Methods Materials Ovalbumin was prepared according to the method of Kekwick and Cannan [8]. Asparaginyl-carbohydrate fractions were isolated according to Huang et al. [9]. The AC-C and AC-D fractions were further subfractionated into AC-CI and -C2, and AC-D~ and -D2 using anion-exchange chromatography in borate buffer [10, 11]. p-Nitrophenyl a-D-mannoside, used for binding experiments, was purchased from P-L Biochemicals and used without further purification. Sugars and other sugar derivatives were purchased from Pfanstiehl. Concanavalin A was prepared by a modification of the m e t h o d reported by Agrawal and Goldstein [12]. Jack bean meal (ICN; 100 g) was extracted overnight with 400 ml of 1.0 M NaC1 at 4°C. The mixture was centrifuged at 8500 rev./min for 15 min at 4°C and the supernatant filtered through cheesecloth. The clear supernatant (approximately 300 ml) was chromatographed on a column (8.5 × 45 cm) of Sephadex G-50 equilibrated with 1 M NaC1. The column was washed with 1 M NaC1 until no more protein could be detected by
358 absorbance at 280 nm. Concanavalin A was eluted with 1 M NaC1 solution containing 0.1 M D-glucose. The solution of concanavalin A was concentrated against PVP to a final protein concentration of approximately 2.5 mg/ml, dialyzed extensively against 0.15 M NaC1, and stored in solution at 5°C. Ovalbumin was fractionated on concanavalin A-Sepharose as previously described [ 11 ].
Immobilization o f concanavalin A Concanavalin A was covalently attached to Sepharose according to the procedure of Porath et al. [13]. Protein concentration on the gel was estimated as 5 mg of concanavalin A per ml of gel by calculating the difference between the amount of concanavalin A in the reaction initially and the uncoupled protein recovered in the wash. The concanavalin A-Sepharose was stored at 4°C in a 0.01 M sodium acetate buffer, pH 5.0, containing 0.15 M NaC1, 1 mM CaC12 and MnCI2, and 0.1% Thimerasol. Identification o f asparaginyl-carbohydrate components o f the ovalbumin fractions U n b o u n d and bound fractions of ovalbumin from concanavalin A-Sepharose chromatography were each digested with pronase and the resulting AC mixtures chromatographed on Dowex 50W-X2 according to Huang et al. [9]. The AC mixtures and the purified, desalted AC-C and AC-D fractions were further chromatographed using anion-exchange chromatography in borate buffer [10, 11]. Digestion o f ovalbumin and A C fractions with a-D-mannosidase-Sepharose Native ovalbumin and its fractions I and II from concanavalin A-Sepharose chromatography, and AC-D were digested with a-D-mannosidase-Sepharose as described by Shepherd and Montgomery [14]. The hydrolyzed mannose was determined using anion-exchange chromatography in borate buffer as described by Lee et al. [10]. Determination o f relative binding capacities o f ovalbumin and AC for concanavalin A at 10°C Relative binding capacities for concanavalin A of ovalbumin and its AC components were determined using inhibition of binding of p-nitrophenyl ~-D-mannoside as described by Bessler et al. [15]. The general procedure involved displacement of a constant known concentration of the chromophore from the binding site on concanavalin A by various concentrations of competing ligands. The release of chromophore was measured spectrophotometrically and binding constants calculated (see Results). All experiments were run in 0.01 M sodium acetate buffer, pH 5.0, with 0.15 M NaC1, 1.0 mM MnC12, and 1.0 mM CaC12, using a Cary 118 recording spectrophotometer with a thermostated cell compartment. Matched divided cells from Hellma with a path length of 8.75 mm were used. Ligands tested for competition with p-nitrophenyl a-D-mannoside were the following: ovalbumin; ovalbumin fractions I and II; ovalbumin treated with a-D-mannosidase (OvalMan); ovalbumin fraction II treated with ~-D-mannosidase (Oval II Man ); AC-A, -B, -C, -C1, -C2,
359
-D, -D1, -D2 and -E; AC-D treated with a - D - m a n n o s i d a s e (AC-DMan); and methyl a-D-mannoside. Concentrations of ovalbumin and its derivatives were deter'e 280nm 1~ = 7.5) and by the phenol/sulfuric acid mined spectrophotometrically ~ m e t h o d [16] for mannose equivalents. All other ligand concentrations were determined by weight and by quantitation of mannose equivalents. Essentially, the only modifications in the m e t h o d of Bessler et al. [15] were the following: (a) the concentration of p-nitrophenyl a-D-mannoside in the cuvette after mixing was 0.05 mM in displacement experiments with competing ligands, and (b) the pH and composition of the buffer was as described above.
Determination o f binding affinities o f ovalbumin and A C-D for concanavalin A at 25°C A comparison of K D values was made for ovalbumin and AC-D binding to concanavalin A at 25°C by the m e t h o d described above at 10°C based on the model by Bessler et al. (using displacement of a constant a m o u n t of p-nitrophenyl ~-D-mannoside) and by the following procedure (using displacement of a series of concentrations of p-nitrophenyl a-D-mannoside by a fixed concentration of competing ligand): a stock solution o f p - n i t r o p h e n y l a-D-mannoside of known concentration (approximately 0.5 mM) was prepared. Aliquots of this solution were pipetted into tubes and buffer was added to a final volume of 2.7 ml, giving final concentrations of p-nitrophenyl a-D-mannoside of 0.05 to 0.25 mM. To each of these solutions was added 0.3 ml of a known concentration of a ligand solution. A series of these experiments.was set up [15], resulting in a set of displacement curves at variable chromophore concentrations and a constant a m o u n t of total ligand. Difference spectra were recorded at 25°C using a Perkin-Elmer 124 recording spectrophotometer. Double reciprocal plots of [PT]/[PD] versus 1/[D] were constructed at each ligand concentration as described by Klotz [17]. The slope of this line is 1/Kapp where KD KaPp - KL[L] + 1 " K D is the association constant o f p - n i t r o p h e n y l a-D-mannoside for concanavalin A, K L is the ligand association constant and [L] is ligand concentration.
Results
Ovalbumin can be separated into two components on concanavalin A-Sepharose [11] to give an u n b o u n d fraction (I), and a bound fraction (II) which was eluted with 0.1 M m e t h y l a-D-mannoside or -glucoside [11]. The ratio of II to I was 2 : 1 with an overall recovery of 85--90%. Ovalbumin fractions I and II contained 4.2 and 2.3 mol of glucosamine per mole of protein, respectively. The binding of ovalbumin fraction II occurred only below 15°C. However, once bound, fraction II could n o t be removed by raising the temperature. The effects of the differing carbohydrate structures on binding were studied by digesting the ovalbumin fractions I and II four times with pronase to obtain the corresponding AC fractions as described previously [9,11]. Fraction II contained primarily AC-C2, -D2, and -E with some AC-A present. Fraction I contained AC-B, -C~, and -D1, again with some AC-A. Ovalbumin fraction II
360 could be further subfractionated on the basis of the differing binding affinities of AC-C2, -D2 and -E for concanavalin A. After elution of ovalbumin fraction I, the column of concanavalin A-Sepharose was developed with a linear gradient up to 0.1 M methyl a-D-glucoside. No definitive separation of peaks occurred, but it was evident that more than one component was present, and that the AC-A, -C2, -D2 and -E fractions in the whole protein were further fractionated.
Determination of relative binding affinities of ovalbumin and AC for concanavalin A Although the concanavalin A-Sepharose column was useful in the study of ovalbumin binding to free concanavalin A and subsequent fractionation of the protein on the basis of microheterogeneity in the carbohydrate, some difficulty was encountered in the study of AC binding to concanavalin A-Sepharose because AC-C2, -D2 and-E were n o t eluted with 0.1 M methyl-a-D-mannoside. It was therefore necessary to use a different technique to compare the relative binding affinities of the whole protein and its isolated carbohydrate for concanavalin A. Bessler et al. [15] described a spectroscopic method for the determination of binding constants for various simple sugar ligands to concanavalin A. This m e t h o d utilizes the blue shift seen when p-nitrophenyl a-D-mannoside binds to protein [18], with a m a x i m u m absorbance change at 317 nm. With certain systems the amounts of free and bound chromophore, [D] and [PD], free and bound ligand, [L] and [PL], and free protein [P] can be calculated from the observed absorbance change, and binding constants for the competing ligands can be determined. The equilibria are represented below: L + P + D.K~PD + L
PL + D.
~PDL
where KD is the association constant for p-nitrophenyl a-D-mannoside, KL is the association constant for the ligand L, KLD is the association constant for the ligand L for the PL complex, and KDL is the association constant for the ligand L for the PD complex. The expression of mass balance (2) can be written: [PT] = [PD] + [P] + [PL] + [PDL]
(2)
Relationship 3 can then be derived from 1 and 2: I
[P_T] i _ 1] = 1 + Kb[L] [PD] + [PDL] J [D] KD[1 +KDL[L]
(3)
By defining the concentrations of free and complexed p-nitrophenyl a-D-mannoside in Eqn. 3 in terms of the experimental data (see Materials and Methods and ref. 15) Eqn. 4 can be written:
I AAmax
~kA
1 + EL[L]
361 For the determination of the value of KDL, data for each value of [L] (approximated by [LT]) were fit by a non-linear least squares program (Nonlin, C.M. Metzler, The Upjohn Company), using values for K L between 120 and 1000 mM-1. According to Bessler et al. [15] the binding of simple sugars to concanavalin A can be represented by a system where only one molecule can bind at a carbohydrate site on the concanavalin A monomer. This assumption eliminates the [PDL] complex, KDL and KLD become very small and Eqn. 3 reduces to Eqn 5: [F[PDI[PT]
11
[D] = 1/KD + ~ [ L ]
(5)
A plot of ([PT]/[PD] -- 1) [D] versus [L], where [L] is approximated by [LT] , should give a straight line with a slope equal to KL/K D and a y-intercept equal to 1/K D. The assumption that [L] = [LT] Can be made when [PL] < < [LT]. Eqn. 5 can be rearranged to form Eqn. 6 in which all the terms on the right side of the equation are constants or can be experimentally determined, and a value for E L can be calculated at each total ligand concentration. [PD] [PT] [PD] KD[D]
KL=
[PD] F
KD[D]L[LT] -- [Pw] +
KD[D-----~[PD] + [PD]]
(6)
This equation allows for the determination of g L without approximating [L] to [LT]. The relative binding affinities of ovalbumin and its AC fractions were compared as competing ligands using Eqns. 4, 5 or 6.
Inhibition by several ovalbumin fractions o f binding o f p-nitrophenyl a-D-mannoside to concanavalin A It was found both in this study and by Bessler et al. [15] that the evaluation of binding constants assuming one binding site on each concanavalin A m o n o m e r is valid for methyl a-D-mannopyranoside, for which a linear plot results. The binding constant so calculated in the present study for this ligand was 1.7 • 104 M -1 at pH 5.0 and 10°C, compared to 1.65 • 104 M -1 at pH 7.0 and 10°C [15]. For methyl a-D-mannoside and other ligands where concentrations necessary for binding are large and binding constants are small, the total a m o u n t of ligand b o u n d is small compared to the total ligand present, and indeed [L] can be approximated by [LT]. Ovalbumin fractions I and II, Oval Man and Oval II Man were used as competing ligands for the binding of p-nitrophenyl a-D-mannoside to concanavalin A at 10°C and pH 5.0. The plots of ([PT]/[PD] -- 1)[D] versus [LT] were linear over the concentrations studied and the fractions I, II and Oval Man showed binding constants of 3.0 • 104 M -1, 2.0 • 106 M -1 and 1.7 • 104 M -I, respectively (Fig. 1). Oval II Man did n o t displace p-nitrophenyl a-D-mannoside from concanavalin A over these same concentrations. The competitive binding data for the native ovalbumin did n o t show a linear relationship when plotted according to the relationship of Eqns. 4, 5 or 6, so
362
0.4--
E
-
a ~
°.2-i
121
O--
°.~J--
OvalTr
i
o/250
/
m
Q
VO.2~
;o°
D
X
X
O~al
0.1-Q
X
v
O-J
0'.2
[LT] (mM)
I
°.'04
I
I 0.02
I
I 0.0,
[LT] (mM)
Fig. 1. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] versus [ L T ] f o r o v a l b u m i n (Oval), o v a l b u m i n f r a c t i o n I ( O v a l I), ovalb u m i n f r a c t i o n II ( O v a l II) a n d o v a l b u m i n t r e a t e d w i t h c~-D-mannoside ( O v a l M) a t 1 0 ° C , a c c o r d i n g t o E q n . 5. T h e t o t a l c o n c e n t r a t i o n o f c o n c a n a v a l i n A ( [ P T ] ) is 0 . 0 3 9 m M in c o m b i n i n g sites, a s s u m i n g a m o n o m e r m o l e c u l a r w e i g h t o f 2 7 0 0 0 . T h e p - n i t r o p h e n y l c~-D-mannoside c o n c e n t r a t i o n is 0 . 0 5 m M a f t e r m i x i n g . O t h e r e x p e r i m e n t a l d e t a i l s are d e s c r i b e d u n d e r M e t h o d s . Fig. 2. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T] f o r o v a l b u m i n a t 2 5 a n d 1 0 ° C . O t h e r c o n d i t i o n s are as d e s c r i b e d in Fig. 1 a n d u n d e r M e t h o d s .
that these three models did not account for the complexity. However, the possibility that there is cooperative interactions of the binding of ovalbumin molecules at the two sites of a concanavalin A dimer is suggested by the linear plot of ([PT]/[PD] -- 1)[D] against [LT] 2 It was known from studies of the binding of ovalbumin to concanavalin A-Sepharose, that negligible interaction occurred at 25°C. The interaction of ovalbumin with concanavalin A in solution was studied at 25°C to determine if the complexity of binding at 10°C could be eliminated by an increase in temperature. The experiments were conducted as described for 10°C. A plot of ( [ P T ] / [ P D ] - 1)[D] versus [LT] could be approximated by a straight line (Fig. 2), and from the slope of this line, a binding constant for ovalbumin at 25°C was calculated to be 1.7 • 10 s M -1. Experiments were also conducted at 25°C in which p-nitrophenyl ~-D-mannoside at differing concentrations was displaced from the concanavalin A by a fixed a m o u n t of ovalbumin. The results were analyzed using the double reciprocal plot described by Klotz [17]. A linear relationship was found at concentrations of ovalbumin up to 0.0158 mM. Ligand binding constants were calculated at each concentration with the following results: at [ L T ] ---- 0.0026 mM, E L = 1 . 5 " l 0 s M - l ; at [ L T ] = 0.0079 mM, K b = 1.6 • 10 s M-'; and, a t [ L T ] = 0 . 0 1 5 8 mM, K L = 0 . 8 " l 0 s M - 1 . T h e plot at low concentrations of ovalbumin gave a y-intercept of n = 1, indicating competitive displacement of p-nitrophenyl ~-D-mannoside from the
363
TABLE I A SUMMARY OF THE BINDING C O N S T A N T S FOR O V A L B U M I N AND ITS D E R I V A T I V E S Ligand
Temp.
K L × 10 -5 (M - I )
(°C)
Method of calculation * 1
Ovalbumin Ovalbumin fraction I O v a l b u m i n f r a c t i o n II Oval Man
10 25 10 10 10
2
3
1.6±0.8 1.70 0.30 20.00 0.17
1.23 -+ 0 . 1 1
* 1~ E q n . 5; 2, eqn. 6; 3, d o u b l e r e c i p r o c a l p l o t s o f [ P T ] / [ P D ] against 1 / [ D ] .
carbohydrate binding site and one to one binding to concanavalin A. The Scatchard plots [19] at low protein concentrations were linear, but plots at higher concentrations (>0.0079 mM) showed deviations from linearity, suggesting that some cooperativity is present also at 25°C. A summary of the binding constants determined for all the ovalbumin fractions examined is shown in Table I.
Inhibition by the AC fractions o f binding p-nitrophenyl a-D-mannoside to concanavalin A The interaction of the isolated ovalbumin AC fractions with concanavalin A was investigated to compare the relative binding affitdties of the AC to the whole protein and to determine which units of the carbohydrate structure were important for binding. The five AC fractions isolated from Dowex-50 chromatography were used as competitive inhibitors of the binding of p-nitrophenyl a-D-mannoside to concanavalin A at 10°C (Fig. 3). As in the case of ovalbumin, all the plots were non-linear over approximately the same concentration range as that used for ovalbumin. Attempts to linearize the data using the models expressed in Eqns. 4, 5 and 6 were again unsuccessful. The use of Eqn. 6 for AC-D, to calculate KL at each total ligand concentration, resulted in values for KL which increased with increasing [LT], ranging from 6.6 • 104 M -1 at [LT] = 0.013 mM, to 1 . 0 . 106M -' at [LT] = 0.038 mM. However, plots of ( [ P T ] / [ P D ] - - 1 ) [ D ] versus [LT] 2 were linear, suggesting some cooperative interaction of the binding of the AC molecules to each unit in the concanavalin A dimer. Fig. 4 shows the results of the binding of AC-D, -D1, -Df, and -D Man to concanavalin A at 10°C. Fig. 5 shows similar plots for the subfractions AC-C, and -C2. AC-D Man and AC-C1 have almost no affinity for concanavalin A, while AC-D2 and AC-C2 are both slightly stronger in their binding than the parent AC-D and -C fractions. At 25°C, as in the case of ovalbumin, the binding of AC-D to concanavalin A became less complex, and plots of ([PT]/[PD] -- 1)[D] versus [LT] (Fig. 6) and the double reciprocal plots were linear. The binding constant from Figure 6 was calculated at 0.7 • 10 s M -1. Binding constants from the double reciprocal plots were as follows: at [LT] = 0.014 raM, KL = 0.6 • l 0 s M-l; at [LT] = 0.028 mM.
364
0.6--
I.S--
D
E
A C
~
~ O , 4 - -
a
q
!
o
°0--
.i o
t"7 o.s-
t"7 o . 2 - O_
13
0._
•t- ×
D1 0.0--
x
0-0--
1 0.00
[
0.02
[LTI
I
l
l
0-04
I
O.OG
I
0-0
(mM)
I
I
0.I
7-
0.2
ILT] (mM] .
.
o
Fig. 3. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T ] f o r t h e a s p a r a g m y l - c a x b o h y d r a t e f r a c t i o n s a t 10 C. O t h e r c o n d i t i o n s axe as d e s c r i b e d in Fig. 1 a n d u n d e r M e t h o d s . Fig. 4. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T ] f o r t h e A C - D s u b f r a c t i o n s at 1 0 ° C . O t h e r c o n d i t i o n s axe as d e s c r i b e d in F i g . I a n d u n d e r M e t h o d s .
0.6--
0.4--
C
E
E 0*4--
r7
~--
J
0.2--
i i
r7
i2s°
0-2--
O. q
0.0--
0.0--
I
0.00
I
I
0.02
[LT]
I
I
0.04
(mM)
I
I
0-08
0.0
I
I
0.02
1
[
0.04
[LT] (raM)
F i g . 5. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [L T] f o r t h e AC-C s u b f r a c t i o n s a t 1 0 ° C . O t h e r c o n d i t i o n s axe as d e s c r i b e d in F i g . 1 a n d u n d e r M e t h o d s . F i g . 6. P l o t o f ( [ P T ] / [ P D ] - - 1 ) [ D ] v e r s u s [ L T ] f o r t h e A C - D s u b f r a c t i o n s a t 2 5 a n d I O ° C . O t h e r c o n d i t i o n s a t e as d e s c r i b e d in F i g . 1 a n d u n d e r M e t h o d s .
365 TABLE
II
A SUMMARY
Ligand
AC-D AC-D]:, A C - D lvian
OF THE BINDING
CONSTANTS
Temp. (°C)
25 10 10
FOR
THE AC-D FRACTIONS
K L X I0 -s ( M -i) M e t h o d of calculation *
1
2
3
0.7 0.25 0.07
1.1±0.2
0.52±0.18
* 1, E q n . 5; 2, e q n . 6; 3, d o u b l e r e c i p r o c a l p l o t s o f [ P T ] / [ P D ]
against 1/[D].
K L = 0.3 • 10 s M-l; and, at [LT] = 0.043 mM, K L = 0.7 • 10 s M -1. Using Eqn. 6 the binding co n s tant at [aT] = 0.028 mM was calculated as 0.9 ± 0.2 • 10 s M-'; and, at [LT] = 0.43, K L = 1.3 ± 0.1 • 10 s M -1. A summary of the binding constants for the AC-D fractions is shown in Table II. Discussion Concanavalin A, covalently attached to Sepharose, has been used as a convenient m e t h o d for studying the interaction o f macromolecules with this lectin. In most instances, the b o u n d protein is eluted from the concanavalin A-Sepharose using 0.1 M m e t h y l a-D-mannoside or -glucoside. Native ovalbumin was separated into two fractions, one which elutes with starting buffer (I), and the o th e r (II) that is eluted with 0.1 M m et hyl a-D-glucoside or -mannoside [ 11]. F u r t h e r fractionation o f fraction II was achieved using a linear gradient o f m e t h y l a-D-mannoside, indicating a heterogeneity of binding species. As will be shown later, the fractions can be related to the microheterogeneity of the c a r b o h y d r a t e groups in the glycoprotein and their differing affinities for concanavalin A. The c a r b o h y d r a t e of ovalbumin has been separated into seven asparaginyl c a r b o h y d r a t e fractions, some of which m ay still be heterogeneous [11]. These fractions may be grouped into three classes: Class I with no non-reducing terminal a-D-mannosyl residues, Class II with bot h a-D-mannosyl and N-acetyl~-D-glucosaminyl non-reducing terminal residues, and Class III with only a-D-mannosyl non-reducing terminal residues. By pronase digestion of the ovalbumin fractions I and II and subsequent isolation of the AC-fractions [11] it was shown that fraction I contained c a r b o h y d r a t e groups that were principally of Class I and fraction II principally of Class III, with some overlap of the Class II c a r b o h y d r a t e groups. Since it was required to com pa r e the binding of ovalbumin and the AC-fractions to concanavalin A it was impossible to use equilibrium dialysis or a concanavalin A-Sepharose column, to which ovalbumin fraction I did n o t bind. A spectroscopic p r o c e d u r e of Bessler et al. [15] was followed in which the competition for the binding to concanavalin A o f p - n i t r o p h e n y l a-D-mannoside and the c a r b o h y d r a t e ligand in question was determined from the difference in the spectrum o f b o u n d and free p - n i t r o p h e n y l a-D-mannoside. The model of Bessler et al. [15] assumes one t o one binding at the c a r b o h y d r a t e site, and for
366 simple sugars, such as methyl a-D-mannoside, this model is valid. However, other types of interactions cannot be ruled out by this procedure and indeed ovalbumin and its AC fractions showed non-linear binding, indicating a greater complexity than the simple model that applied to methyl a-D-mannopyranoside. Several workers have suggested that other non-carbohydrate interactions might be involved in the binding of ligands to concanavalin A. Human interferon apparently binds to concanavalin A-Sepharose through both carbohydrate and non-polar interactions [6], and binding of concanavalin A to fat cells is through multiple binding sites, stabilized by both carbohydrate and hydrophobic forces [ 7]. The competitive inhibition data for ovalbumin and its AC fractions when expressed assuming the model of Bessler showed an initial slope that progressively increased as the competing ligand concentration increased. In those cases where there was a high affinity binding, such as AC-D2 or ovalbumin fraction II the lower affinity at small ligand concentrations was not measurable or was less apparent. Similarly, under the conditions of the experiment, ligands with little or no affinity for concanavalin A, such as ovalbumin fraction I, AC-C1, or AC-D~ do not show the non-linear plot. Ovalbumin, containing a mixture of at least seven glycoproteins, presents a curved plot on this Bessler model, as a result of the two extremes of binding. The curvature in the plot (Fig. 1) is probably n o t simply the result of a mixture of binding affinities at the carbohydrate site since presumably the tightest binding component would bind first, giving a plot curving in the opposite manner to that observed. Additionally, it is clear from the change in binding with concanavalin A at two temperatures, 10 and 25°C, that the conformations of the proteins, aside from the carbohydrate ligand, play a role in the nature of the complexation. Since the spectroscopic procedure of Bessler et al. can only detect the competitive displacement of the p-nitrophenyl a-D-mannoside from the carbohydrate binding site on each m o n o m e r unit, it is only by indirect evidence that non-carbohydrate binding or cooperative interactions are indicated. Such is the case however for ovalbumin and its AC fractions since the data show a linear relationship between ( [PT] / [ PD] -- 1) [D] and [ L w ] 2. A more detailed description of this cooperative binding at the two sites of the concanavalin A dimer requires a different experimental approach, such as equilibrium dialysis for the AC fractions, and the means to prepare pure glycopeptides, which is presently possible in a relatively few cases. Although quantitative binding constants were n o t obtained for all of the fractions studied, relative binding affinities could be established on the basis of concentrations of ligand necessary to displace 50% of the bound p-nitrophenyl a-D.mannoside, analogous to the methods of calculation using dextran binding inhibition [20,21], hemagglutination inhibition [22], and inhibition of binding of '2SI-concanavalin A to erythrocytes [23]. Although these methods may not result in accurate binding constants, an approximation of relative affinities can be obtained. Table III shows the 50% inhibition concentration for the ovalbumin and AC fractions. On the basis of concentrations of AC fractions necessary for p-nitrophenyl a-D-mannoside displacement, the fractions are collected into four groups from the relative binding affinities: Group 1 (AC-D2 and AC-C2), which has an affinity for concanavalin A approximately 8--9 times
367 TABLE III RELATIVE
BINDING CAPACITIES
Ligand
Ovalbumin Ovalbumin fraction I Ovalbumin fraction II Oval Man
OF OVALBUMIN
Conc. to give
Relative binding
50% i n h i b i t i o n (M × 1 0 - s )
capacity *
3.0
AC.C1 AC-C2 AC-C AC-D 1 AC-D 2 AC-D AC-D Man AC-E M e t h y l cx-D-manno side
TO Con A
5.3 2 . 0 ** 160.0 1 . 2 **
0.1
Oval II Man AC-A AC-B
AND AC FRACTIONS
*** 3.0 4.5
5.3 3.6 *** 8.0 5.3 2 . 5 ** 8.9 8.0 1 . 0 **
2.0 3.0 1.8 2.0 2.2 16.0
7.3 1.0
* R e l a t i v e t o t h e c o n c e n t r a t i o n o f m e t h y l a - D - m a n n o s i d e t o g i v e 5 0 % i n h i b i t i o n = 1.0. ** C a l c u l a t e d f r o m a r a t i o o f KL/KM, w h e r e K M = t h e a s s o c i a t i o n c o n s t a n t f o r m e t h y l - a - D - m a n n o s i d e . ** * F o r t h e c o n c e n t r a t i o n s t u d i e d n o b i n d i n g w a s o b s e r v e d .
greater than m e t h y l a-D-mannoside; Group 2 (AC-A and AC-E) with an affinity 5--7 times greater; Group 3 (AC-B, AC-D1 and AC-D Man) about the same as m e t h y l a-D-mannoside; and, Group 4 (AC-C1) which shows less binding than the monosaccharide. The structures within these fractions that are binding to concanavalin A might be predicted on the basis of model c o m p o u n d studies and known structural features of the AC components [11,24]. Goldstein and co-workers [25,26] have investigated the binding of several mannose-conraining di- and trisaccharides, as shown below relative to methyl a-D-mannoside: methyl a-D-mannoside Gal-a-(1->6)-Man-a-(I->2 )-Man Mama-(1->2 )-Man Gal-a-(1->2 )-Man-a-(I->2 )-Man Man-a-(1-~2 )-Man-a-(I->2 )-Man
1.0 1.2 1.9 2.1 10.0
AC-C1, from which no a-D-mannosyl residues were hydrolyzed by a-D-mannosidase and no c/s-hydroxyl groups on the internal mannose residues complex with borate [11], shows a binding to concanavalin A less than that for methyl a-D-mannopyranoside. AC-B, even with no terminal a-D-mannosyl residues, shows binding equivalent to m e t h y l a-D-mannopyranoside and is probably due to internal 1 -~ 2 linked a-D-mannosyl residues, such as proposed earlier, where AC-B is derived from AC-D2 by the addition of N-acetylglucosaminyl residues on each terminal mannose [11]. This is also consistent with the weak complexation of AC-B with borate [11]. Similarly, the binding of AC-D Man is due to the residual AC-D1, from which only one mannosyl residue is hydrolyzed by a-D-mannosidase [ 11].
368 The tight binding of AC-E and AC-D2 agrees with the fact that each oligosaccharide has three terminal a-D-mannosyl residues, the addition affinity in AC-D2 being due to the internal 1 -~ 2 linked mannosyl residue [24]. The correlation between the complexation with concanavalin A and borate is again striking. The intermediate binding affinity of Group 2 compounds, AC-A and AC-C2, would be predicted by the presence in each of a terminal and a 1 -~ 2 linked a-D-mannosyl residue. The order of relative binding affinities for the ovalbumin fractions from Table III is as follows: Ovalbumin fraction II > ovalbumin > ovalbumin fraction I > O v a l Man ~ Oval II Man. Fraction I, which is not retained on a column of concanavalin A-Sepharose, does bind to concanavalin A in solution; apparently the binding is not tight enough to retard this component on the concanavalin A-Sepharose column. As expected, the ovalbumin fraction II, which binds to immobilized concanavalin A, also binds tightly to concanavalin A in solution. It will be noted (Table III) that ovalbumin fraction II, which contains the carbohydrate groups AC-D2 and AC-E, has a binding capacity that is about 20 times greater than the asparaginyl-carbohydrates. It is proposed that the concanavalin A and ovalbumin proteins interact through non-carbohydrate sites thus statistically increasing the probability of the a-D-mannosyl residues in fraction II binding with the concanavalin A carbohydrate binding site. Competition with the p-nitrophenyl a-D-mannoside would thus appear to be increased over that of the AC-D2 or AC-E, which do not have the same noncarbohydrate interactions. It has been shown [14] that 40% of the total mannose in the isolated AC can be removed enzymatically, but only one-half of this value or 20% of the total mannose residues, can be hydrolyzed from native ovalbumin. Following the hydrolysis of ovalbumin fraction II by a-D-mannosidase, no binding to concanavalin A was observed over the concentrations studied. Apparently, only the peripheral mannose residues in this protein are important for and accessible to interaction with both concanavalin A and a-D-mannosidase, with the core region perhaps " b u r i e d " in a portion of the polypeptide structure. This result implies that the inner " c o r e " region contributes little or no binding in the interaction of the ovalbumin glycopeptide with concanavalin A. Native ovalbumin and its AC fractions bind with approximately the same affinity (Figs. 1 and 3). Thus the carbohydrate residues which interact with concanavalin A are equally exposed in the whole protein and the isolated AC. This is in agreement with the finding of Smith et al. [22] who found ovalbumin and its glycopeptide equally effective in inhibiting hemagglutination by concanavalin A. In contrast, Young and Leon [21] found that the glycopeptide mixture inhibited dextran precipitation at concentrations ten-fold less than the whole protein. In both cases, however, comparisons with the present study are difficult since methods for and conditions of measurement of binding were different. In comparing the results of studies on the binding of ligands to concanavalin A it is important to consider the system being used. Kornfeld and Ferris [23] looked at the inhibition by IgG glycopeptides of the binding of concanavalin A to cell surfaces. This type of binding is very complex [7] and probably involves the binding of concanavalin A to the cells through non-carbohydrate as
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